Contents

Introduction

These protocols are used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid. It utilizes a fast high-fidelity non-displacing DNA polymerase (Phusion) to replicate the plasmid including the desired mutation. DpnI then cuts up all your old plasmid. While these protocols can make reliable controlled mutations, the constraints may not work for you; check out the consensus protocol for more options.

Duplex Oligo Mutation

This protocol uses two oligos, each containing the desired mutation. It is more reliable than the other protocol, but can only mutate at one location (1-3bp) at a time.

Method

1. Design mutagenesis primers.

The targeted mutation should be included into both primers.

The mutation can be as close as 4 bases from the 5-terminus.

The mutation should be at least 8 bases from the 3-terminus.

At least eight non-overlapping bases should be introduced at the 3-end of each primer.

references

Single Oligo Mutation

This protocol only uses one oligo per mutation site (cuts the oligo cost in half) but can mutate more than one site at a time. While this protocol is less reliable than the previous protocol (only ~25% of colonies will contain the desired mutation(s)), if you're doing multiple mutations on the same piece of DNA than this can save you many days of work by just sequencing a few more colonies.